Conversion of cholesterol to pregnenolone is the first and rate-limiting step in the synthesis of all steroid hormones. This conversion entails three steps: 20-hydroxylation, 22-hydroxylation, and cleavage of the C.sub.20 -C.sub.22 bond to produce pregnenolone and isocaproic acid. These three steps are mediated by a single mitochondrial cytochrome, formerly known as 20,22-desmolase and now identified as P450scc. P450scc is the cholesterol-specific terminal oxidase in a mitochondrial electron transport system consisting of a flavoprotein (adrenodoxin reductase), an iron-sulfur protein (adrenodoxin), and cytochrome P450scc. Deficient P450scc activity causes lipoid adrenal hyperplasia, a generally lethal disease.
There are a number of related cytochrome P450 enzymes. Microsomal cytochrome P450's catalyze a wide variety of oxidation reactions of endogenous substrates as well as xenobiotics, including drugs. On the contrary, mitochondrial cytochrome P450's show no known enzyme activities to xenobiotics.
The molecular cloning and nucleotide sequence of cDNA for mitochondrial cytochrome P450scc of bovine adrenal cortex has been reported. However, the corresponding cDNA, mRNA, chromosomal DNA, and/or primary amino acid sequence of human mitochondrial cytochrome P450scc was not known prior to the present invention. Bovine mitochondrial cytochrome P450scc has not been utilized in humans, at least in part because of its unknown activity and potential antigenicity in humans as a result of its different primary amino acid sequence.
Accordingly, there is need for techniques of preparing human mitochondrial cytochrome P450scc (preferably by techniques of genetic engineering), and for developing its use in reducing cholesterol levels in humans.